Team:Marburg SYNMIKRO/Results

From 2012.igem.org

(Difference between revisions)
(Biobricks:)
(Biobricks:)
 
(28 intermediate revisions not shown)
Line 4: Line 4:
<div class="conboxb">
<div class="conboxb">
</html>
</html>
-
== <center>'''Results'''</center> ==
 
-
 
-
 
=== '''Biobricks:''' ===
=== '''Biobricks:''' ===
-
 
-
 
'''[http://partsregistry.org/Part:BBa_K866000 BBa_K866000] (CFP-Fusion Brick):'''
'''[http://partsregistry.org/Part:BBa_K866000 BBa_K866000] (CFP-Fusion Brick):'''
-
In order to evaluate putative recombination events in E. coli, we chose to generate fluorescent fusion proteins which are to be localized at different compartments within a cell.  
+
In order to evaluate putative recombination events in ''E. coli'', we decided to generate fluorescent fusion proteins, which localize at different compartments within a cell.  
-
[http://partsregistry.org/Part:BBa_E0020 BBa_E0020] is a biobrick coding for the Cyan Fluorescent Protein (CFP). To build a CFP suitable for fusion proteins, we chose this biobrick as a template to create a CFP coding sequence (CDS) which can be cloned in-frame to other protein coding sequences via [http://partsregistry.org/Assembly:Standard_assembly Standard Assembly].  
+
[http://partsregistry.org/Part:BBa_E0020 BBa_E0020] is a biobrick coding for the Cyan Fluorescent Protein (CFP). To make CFP suitable for fusion proteins, we used this biobrick as a template to create a CFP coding sequence (CDS) which can be cloned in-frame to other protein coding sequences via [http://partsregistry.org/Assembly:Standard_assembly Standard Assembly].  
-
The CFP-Fusion Brick was constructed by site directed mutagenesis through polymerase chain reaction (PCR) using primer [https://2012.igem.org/Team:Marburg_SYNMIKRO/Primer MS_16] and [https://2012.igem.org/Team:Marburg_SYNMIKRO/Primer MS_17]. PCR products were separated by agarose gelelectrophoresis, gel extracted and cloned into [http://partsregistry.org/Part:pSB1C3 pSB1C3] via [http://partsregistry.org/Help:Standards/Assembly/RFC10 Standard Assembly]. Positive clones were verified by [[Team:Marburg_SYNMIKRO/Notebook#27.08.2012|colony PCR]] and [http://partsregistry.org/cgi/partsdb/dna.cgi?part_name=BBa_K866000 sequencing]. This fusion brick was later on screened for expression and localization in ''E. coli'' as a synthesized fusion protein with A-modules [[Team:Marburg_SYNMIKRO/Project#Localization domains|GroES and AmiC]] using [[Team:Marburg_SYNMIKRO/Results#Fluorescence microscopy:|fluorescence microscopy]].
+
Our novel CFP-fusion brick was constructed by site directed mutagenesis through polymerase chain reaction (PCR) using primer [https://2012.igem.org/Team:Marburg_SYNMIKRO/Primer MS_16] and [https://2012.igem.org/Team:Marburg_SYNMIKRO/Primer MS_17]. PCR products were separated by agarose gel electrophoresis, extracted from the gel and cloned into [http://partsregistry.org/Part:pSB1C3 pSB1C3] via [http://partsregistry.org/Help:Standards/Assembly/RFC10 Standard Assembly]. Positive clones were verified by [[Team:Marburg_SYNMIKRO/Notebook#27.08.2012|colony PCR]] and [http://partsregistry.org/cgi/partsdb/dna.cgi?part_name=BBa_K866000 sequencing]. The CFP fusion brick was verified by expression and localization in ''E. coli'' as fusion protein with different intracellular localization domains (A-modules) [[Team:Marburg_SYNMIKRO/Project#Localization domains|GroES and AmiC]] using [[Team:Marburg_SYNMIKRO/Results#Fluorescence microscopy:|fluorescence microscopy]].
'''[http://partsregistry.org/Part:BBa_K866001 BBa_K866001] (mRFP-Fusion Brick):'''
'''[http://partsregistry.org/Part:BBa_K866001 BBa_K866001] (mRFP-Fusion Brick):'''
-
Besides a CFP-fusion protein, a monomeric Red Fluorescent Protein was contructed from [http://partsregistry.org/Part:BBa_E1010 BBa_E1010] as template. Site directed mutagenesis was performed via PCR using primer [https://2012.igem.org/Team:Marburg_SYNMIKRO/Primer MS14] and [https://2012.igem.org/Team:Marburg_SYNMIKRO/Primer MS15]. PCR products were separated by agarose gelelectrophoresis, gel extracted and cloned into [http://partsregistry.org/Part:pSB1C3 pSB1C3] via [http://partsregistry.org/Help:Standards/Assembly/RFC10 Standard Assembly]. Positive clones have been verified by colony PCR [link colony-pcr-bild in lab-book?] and sequencing [link sequenzierung in parts registry?].
+
In addition to the CFP-fusion protein, fusion-competent monomeric Red Fluorescent Protein (mRFP) was contructed from [http://partsregistry.org/Part:BBa_E1010 BBa_E1010] as template. Site directed mutagenesis was performed via PCR using primer [https://2012.igem.org/Team:Marburg_SYNMIKRO/Primer MS14] and [https://2012.igem.org/Team:Marburg_SYNMIKRO/Primer MS15]. PCR products were separated by agarose gel electrophoresis, extracted from the gel and cloned into [http://partsregistry.org/Part:pSB1C3 pSB1C3] via [http://partsregistry.org/Help:Standards/Assembly/RFC10 Standard Assembly]. Positive clones have been verified by [[Team:Marburg_SYNMIKRO/Notebook#27.08.2012|colony PCR]] and [http://partsregistry.org/cgi/partsdb/dna.cgi?part_name=BBa%20K866001 DNA sequencing].
-
BBa_K866002 [Link] (G-segment Invertase):
 
-
The G-segment Invertase (Gin) from the phage Mu [Link?] is a protein which can either cause inversions of gene segments flanked by gix-sites [link paper oder Gene bank, da keine sequenz in parts registry?] as inverted repeats or cause deletions of gene segments flanked by gix-sites as direct repeats [link paper bzw. ,,recombination”-grundlagen in unserem wiki?]. Recombination events are facilitated by the Enhancer DANN segments located in cis [link paper?] (see also poste/project [link]).
 
-
We were aiming at constructing a biobrick that codes for Gin [link] and contains a ribosomal binding site (RBS) attached/flanked upstream from it’s CDS.
 
-
Hence, we designed primer (MS11 and MS12 [link]) which result in amplification of Gin containing the well characterized and strong RBS BBa_J61100 [link registry] from the Anderson RBS-family. As template for PCR, chromosomal DNA from E. coli strain C600 Mucts62 (lysogenic for temperature sensitive phage Mu cts62) was used. PCR amplicons were separated via agarose gelelectrophoresis and cloned into pSB1C3 through Standard Assembly [link]. Positive clones were evaluated by colony-PCR [link labbook] and sequencing [link parts registry???].
+
'''[http://partsregistry.org/Part:BBa_K866002 BBa_K866002] (Gin DNA Invertase):'''
-
The G-Segment Invertase Biobrick [link] can be tested with different promoters in order to evaluate the optimal level of expression for recombination events in E. coli to occur.
+
-
BBa_K866002 [Link] (SacB Suicide Gene):
+
The G-segment Invertase (Gin) from [[Team:Marburg_SYNMIKRO/Project#Phage Mu|phage Mu]] is a site-specific DNA recombinase, which causes inversion of gene segments flanked by [[Team:Marburg_SYNMIKRO/Project#Gin recombination|gix sites]] as inverted repeats. If the gene segment is flanked by gix-sites as direct repeats Gin recombinase causes [[Team:Marburg_SYNMIKRO/Project#Site-specific recombination|deletions]] of the gene segment. Efficient recombination depends on a short DNA-segment which acts as recombinational [[Team:Marburg_SYNMIKRO/Project#Enhancer|Enhancer]].
-
In order to select for recombination events in E. coli cells in a culture, the suicide gene SacB [link] was chosen. In our final recombination construct [link project], it shall be located between A and B-modules, causing a deletion of this suicide gene and thus select for positive recombination events.
+
To facilitate expression of Gin the biobrick contains a ribosomal binding site (RBS) attached upstream of the Gin start codon.
-
It codes for a levan sucrase, which can polymerize sucrose from growth media within Gram negative cells to levan, thus disturbing the cell’s metabolism and hence growth, leading to growth inhibition up to cell death [link paper?].
+
-
SacB was amplified using primer MS26 and MS27 [link primer liste] from the suicide plasmid pNTPSXY via PCR. PCR products were separated by agarose gelelectrophoresis, gel extracted and cloned into pSB1C3 [link] via Standard Assembly [link]. Positive clones have been verified by colony PCR [link colony-pcr-bild in lab-book?] and sequencing [link sequenzierung in parts registry?].
+
-
BBa_K866002 [link] can be fused to promoters of different strength and tested for optimal screening through cell death on sucrose rich media.
+
-
=== '''Experiments:''' ===
+
We designed primer [https://2012.igem.org/Team:Marburg_SYNMIKRO/Primer MS11] and [https://2012.igem.org/Team:Marburg_SYNMIKRO/Primer MS12] to fuse the well characterized and strong RBS [http://partsregistry.org/Part:BBa_J61100 BBa_J61100] from the Anderson RBS-family during amplification of the Gin coding sequence. As template for PCR, chromosomal DNA from ''E. coli'' strain C600 Mucts62 (lysogenic for temperature sensitive phage Mu cts62) was used. PCR amplicons were separated via agarose gel electrophoresis and cloned into [http://partsregistry.org/Part:pSB1C3 pSB1C3] through [http://partsregistry.org/Help:Standards/Assembly/RFC10 Standard Assembly]. Positive clones were evaluated by [[Team:Marburg_SYNMIKRO/Notebook#20.09.2012|restriction enzyme digest]] and [http://partsregistry.org/cgi/partsdb/dna.cgi?part_name=BBa%20K866002 DNA sequencing].
 +
The [http://partsregistry.org/Part:BBa_K866002 Gin DNA Invertase Biobrick] can be used with promoters of different strength in order to evaluate the optimal level of Gin-expression to stimulate recombination events in ''E. coli''.
-
==== Generation of test constructs: ====
+
'''[http://partsregistry.org/Part:BBa_K866003 BBa_K866003] (SacB Suicide Gene):'''
-
As a first step towards our complete recombination construct [link poster, bzw. Project], we aimed at generating the putative recombination gene products, meaning the fusion of a complete A-module with a complete B-module with a gix-site in between. These putative recombination constructs would be the outcome as if a recombination event would have had occurred in the complete construct [zu viel “construct” -> alternatives wort???].
+
-
For this purpose, all three complete (HU nicht dabei, trotzdem alle 3 erwähnen?) A-Modules, HU [link], AmiC[link] and GroES[link] have been synthesized by Gene Art (life technologies TM). These A Modules consist each of one localization domain (AmiC, GroES or HU) with the RBS BBa_J61100 [link] and the strong constitutive promotor BBa_J23108 [link] located upstream. Downstream of localization domains, a gix site [link]  is located. Between promotor, RBS, localization domain and the gix site, the scar sequence from the standard assembly [link scar] was included in order to test for the correct reading frame as it would have been necessary for the complete recombination construct. The complete A-modules are flanked by the standard prefix and suffix.
+
-
The complete A-modules coding for AmiC and GroES were cloned N-terminally to
+
-
our fluorescent fusion protein sequences BBa_K866000 (CFP-Fusion Brick) [link],  BBa_K866001 (mRFP-Fusion brick) [link] aswell as the biobrick BBa_K125500 (GFP-Fusion Brick) [link] via standard assembly [Link]. Verification of fluorence was performed via fluorescence microscopy.
+
-
==== Fluorescence microscopy: ====
+
To allow counter-selection against non-recombined plasmids, the suicide gene ''sacB'' was chosen. In the presence of sucrose in the medium, expression of the ''Bacillus subtilis sacB'' gene is toxic for gram negative ''E. coli'' cells. ''sacB'' codes for the enzyme levansucrase, which uses sucrose to produce the polymeric <html>levan (2,6-&beta;-D-fructosyl)<sub>n</sub></html> in the periplasm, which disturbs cell metabolism and growth, leading to cell death. In our final [[Team:Marburg_SYNMIKRO/Project#Construct|recombination construct]], ''sacB'' will be located between the A and B-modules. Successful recombination will cause deletion of the suicide gene and thus cells that contain plasmids with positive recombination events can be selected on sucrose containing media.
-
In order screen for E. coli containing and expressing our test constructs, positively selected colonies through antibiotic restistance have been subjected to fluorescence microscopy.
+
The ''sacB'' gene was amplified using primer [https://2012.igem.org/Team:Marburg_SYNMIKRO/Primer MS26] and [https://2012.igem.org/Team:Marburg_SYNMIKRO/Primer MS27] from the suicide plasmid pNTPS (kindly provided by K. Thormann, MPI Marburg) via PCR. PCR products were separated by agarose gel electrophoresis, extracted from the gel and cloned into [http://partsregistry.org/Part:pSB1C3 pSB1C3] via [http://partsregistry.org/Help:Standards/Assembly/RFC10 Standard Assembly]. Positive clones were verified by [[Team:Marburg_SYNMIKRO/Notebook#17.09.2012|restriction enzyme digest]] and [http://partsregistry.org/cgi/partsdb/dna.cgi?part_name=BBa%20K866003 DNA sequencing].
-
For this purpose, colonies growing on LB-Agar supplemented with antibiotics for BBa_K866000 (CFP-Fusion Brick) [link],   BBa_K866001 (mRFP-Fusion brick) [link] and BBa_K125500 (GFP-Fusion Brick) [link], respectively, were cultured over-night in LB-medium with antibiotics. Small aliquots (10 µL) of overnight cultures were added onto  2% agarose-pads [link protokoll, falls überhaupt vorhanden?] located on microscope slides, covered by cover-slips (doppelt gemoppelt oO) and used for fluorescence microscopy.
+
[http://partsregistry.org/Part:BBa_K866003 BBa_K866003] can be fused to promoters of different strength and tested for optimal counter-selection through cell death on sucrose containing media.
-
Alltogether, 6 different constructs have been observed for fluorescence, being AmiC-GFP, AmiC-CFP, AmiC-mRFP, GroES-GFP, GroES-CFP and GroES-mRFP.
+
<html>
-
As visible in figure XY, fluorescence occurs in cell carrying each construct. Especially in cells expressing GroES-GFP and AmiC-GFP figure XY, intense fluorescence is detectable at cell poles and in the periplasm in contrast to other cell compartments. Similar effects are also visible for GroES-mRFP and AmiC-mRFP, respectively. For GroES-CFP and AmiC-CFP figure XY, fluorescence is detectable, yet no precise localization can be determined.
+
</div>
 +
</html>
 +
<html>
 +
<div class="conboxb">
 +
</html>
-
As a result, our test constructs mimicking (???) a putative recombination event of our planned complete recombination construct [link projet] are expressed in E.coli. Furthermore, GFP- and mRFP fused A-modules show enhanced fluorescence at expected cell compartments, being cell poles for GroES and periplasm for AmiC.
+
=== '''Experiments:''' ===
 +
==== Generation of test constructs: ====
 +
As a first step towards our complete [[Team:Marburg_SYNMIKRO/Project#Construct|recombination construct]], we generated the final recombination gene products i.e. the fusion of a complete A-module with a complete B-module connected by a ''gix'' recombination site. These putative recombination constructs would be the outcome if a recombination event would have occurred in the complete [[Team:Marburg_SYNMIKRO/Project#Construct|recombination construct]].
 +
For this purpose, all three cellular localization domains (A-Modules), [[Team:Marburg_SYNMIKRO/Project#Localization domains|HU, GroES and AmiC]] have been synthesized. The A modules each consist of the localization domain (AmiC, GroES or HU) preceded by a standard ribosome binding site (RBS) [http://partsregistry.org/Part:BBa_J61100 BBa_J61100] and the strong constitutive promotor [http://partsregistry.org/Part:BBa_J23108 BBa_J23108]. Downstream of the localization domains, a [[Team:Marburg_SYNMIKRO/Project#Gin recombination|''gix'' site]] is located. Between promotor, RBS, localization domain and the gix site, the [http://partsregistry.org/Help:Standards/Assembly/RFC10 scar sequence] from standard assembly was included to test for the correct expression of the reading frame. The complete A-modules are flanked by the biobrick standard assembly [http://partsregistry.org/Help:Standards/Assembly/RFC10 RFC10] prefix and suffix.
 +
The complete A-modules coding for AmiC and GroES were cloned N-terminally to
 +
our fluorescent fusion protein sequences [http://partsregistry.org/Part:BBa_K866000 BBa_K866000] (CFP-Fusion Brick),  [http://partsregistry.org/Part:BBa_K866001 BBa_K866001] (mRFP-Fusion brick as well as the biobrick [http://partsregistry.org/Part:BBa_K125500 BBa_K125500] (GFP-Fusion Brick) via standard assembly. Verification of fluorescence was performed via fluorescence microscopy.
-
Discussion:
+
==== Fluorescence microscopy: ====
-
 
+
In order to screen for fluorescent ''E. coli'' harboring our putative test constructs, positively selected colonies through antibiotic resistance have been subjected to fluorescence microscopy.
-
 
+
For this purpose, colonies growing on LB-Agar supplemented with antibiotics for [http://partsregistry.org/Part:BBa_K866000 BBa_K866000] (CFP-Fusion Brick),  [http://partsregistry.org/Part:BBa_K866001 BBa_K866001] (mRFP-Fusion brick) and [http://partsregistry.org/Part:BBa_K125500 BBa_K125500] (GFP-Fusion Brick), respectively, were cultured over-night in LB-medium with appropriate antibiotics.  
-
 
+
Small aliquots (10 µL) of overnight cultures were added onto  2% agarose-pads casted on microscope slides, covered by cover-slips and used for fluorescence microscopy.
-
 
+
-
Outlook:
+
-
 
+
-
 
+
-
 
+
-
[[File:Syn_MR_AmiC-mRFP.jpg|center|450px]]
+
-
 
+
-
 
+
-
[[File:Syn_MR_AmiC-GFP.jpg|center|450px]]
+
-
 
+
-
 
+
-
[[File:Syn_MR_AmiC-CFP.jpg|center|450px]]
+
-
 
+
-
 
+
-
[[File:Syn_MR_GroES-mRFP.jpg|center|450px]]
+
 +
Altogether, 6 different constructs have been analyzed for fluorescence, being AmiC-GFP, AmiC-CFP, AmiC-mRFP, GroES-GFP, GroES-CFP and GroES-mRFP.
 +
As can be seen in Figure 1, fluorescence was observed in all cells carrying a fusion construct. Especially in cells expressing GroES-GFP and AmiC-GFP (Fig. 1 E and B), intense fluorescence was detectable at cell poles for GroES and in the periplasm for AmiC. Similar localizations were also observed for GroES-mRFP and AmiC-mRFP (Fig. 1 D and A). For GroES-CFP and AmiC-CFP (Fig. 1 F and C), fluorescence is detectable, yet for AmiC-CFP no precise localization could be determined.
-
[[File:Syn_MR_GroES-GFP.jpg|center|450px]]
+
As a result, our test constructs representing a putative recombination event of our planned [[Team:Marburg_SYNMIKRO/Project#Construct|recombination construct]] were successfully expressed in ''E.coli''. Furthermore, GFP- and mRFP fused A-modules show enhanced fluorescence at cell poles for GroES and in the periplasm for AmiC.
-
[[File:Syn_MR_GroES-CFP.jpg|center|450px]]
 
 +
[[File:Syn_8.jpg|center|600px]]
-
[[File:Syn_MR_Kontrolle_(P-RBS).jpg|center|450px]]
 
 +
[[File:Syn_MR_Kontrolle_(P-RBS).jpg|center|600px]]
 +
'''Fig. 1''': Fluorescent ''E. coli'' cells harbouring the fusion constructs of [[Team:Marburg_SYNMIKRO/Project#Construct|A- and B-modules]]. '''A''': AmiC-mRFP. '''B''': AmiC-GFP. '''C''': AmiC-CFP. '''D''': GroES-mRFP. '''E''': GroES-GFP. '''F''': GroES-CFP. '''G''': ''E. coli'' harboring a Promotor-RBS construct which serves as a non-fluorescent control. All images were taken by fluorescence microscopy at a magnification of x100.
<html>
<html>

Latest revision as of 00:22, 27 September 2012

Main Page

Contents

Biobricks:

[http://partsregistry.org/Part:BBa_K866000 BBa_K866000] (CFP-Fusion Brick):

In order to evaluate putative recombination events in E. coli, we decided to generate fluorescent fusion proteins, which localize at different compartments within a cell. [http://partsregistry.org/Part:BBa_E0020 BBa_E0020] is a biobrick coding for the Cyan Fluorescent Protein (CFP). To make CFP suitable for fusion proteins, we used this biobrick as a template to create a CFP coding sequence (CDS) which can be cloned in-frame to other protein coding sequences via [http://partsregistry.org/Assembly:Standard_assembly Standard Assembly].

Our novel CFP-fusion brick was constructed by site directed mutagenesis through polymerase chain reaction (PCR) using primer MS_16 and MS_17. PCR products were separated by agarose gel electrophoresis, extracted from the gel and cloned into [http://partsregistry.org/Part:pSB1C3 pSB1C3] via [http://partsregistry.org/Help:Standards/Assembly/RFC10 Standard Assembly]. Positive clones were verified by colony PCR and [http://partsregistry.org/cgi/partsdb/dna.cgi?part_name=BBa_K866000 sequencing]. The CFP fusion brick was verified by expression and localization in E. coli as fusion protein with different intracellular localization domains (A-modules) GroES and AmiC using fluorescence microscopy.


[http://partsregistry.org/Part:BBa_K866001 BBa_K866001] (mRFP-Fusion Brick):

In addition to the CFP-fusion protein, fusion-competent monomeric Red Fluorescent Protein (mRFP) was contructed from [http://partsregistry.org/Part:BBa_E1010 BBa_E1010] as template. Site directed mutagenesis was performed via PCR using primer MS14 and MS15. PCR products were separated by agarose gel electrophoresis, extracted from the gel and cloned into [http://partsregistry.org/Part:pSB1C3 pSB1C3] via [http://partsregistry.org/Help:Standards/Assembly/RFC10 Standard Assembly]. Positive clones have been verified by colony PCR and [http://partsregistry.org/cgi/partsdb/dna.cgi?part_name=BBa%20K866001 DNA sequencing].


[http://partsregistry.org/Part:BBa_K866002 BBa_K866002] (Gin DNA Invertase):

The G-segment Invertase (Gin) from phage Mu is a site-specific DNA recombinase, which causes inversion of gene segments flanked by gix sites as inverted repeats. If the gene segment is flanked by gix-sites as direct repeats Gin recombinase causes deletions of the gene segment. Efficient recombination depends on a short DNA-segment which acts as recombinational Enhancer. To facilitate expression of Gin the biobrick contains a ribosomal binding site (RBS) attached upstream of the Gin start codon.

We designed primer MS11 and MS12 to fuse the well characterized and strong RBS [http://partsregistry.org/Part:BBa_J61100 BBa_J61100] from the Anderson RBS-family during amplification of the Gin coding sequence. As template for PCR, chromosomal DNA from E. coli strain C600 Mucts62 (lysogenic for temperature sensitive phage Mu cts62) was used. PCR amplicons were separated via agarose gel electrophoresis and cloned into [http://partsregistry.org/Part:pSB1C3 pSB1C3] through [http://partsregistry.org/Help:Standards/Assembly/RFC10 Standard Assembly]. Positive clones were evaluated by restriction enzyme digest and [http://partsregistry.org/cgi/partsdb/dna.cgi?part_name=BBa%20K866002 DNA sequencing]. The [http://partsregistry.org/Part:BBa_K866002 Gin DNA Invertase Biobrick] can be used with promoters of different strength in order to evaluate the optimal level of Gin-expression to stimulate recombination events in E. coli.


[http://partsregistry.org/Part:BBa_K866003 BBa_K866003] (SacB Suicide Gene):

To allow counter-selection against non-recombined plasmids, the suicide gene sacB was chosen. In the presence of sucrose in the medium, expression of the Bacillus subtilis sacB gene is toxic for gram negative E. coli cells. sacB codes for the enzyme levansucrase, which uses sucrose to produce the polymeric levan (2,6-β-D-fructosyl)n in the periplasm, which disturbs cell metabolism and growth, leading to cell death. In our final recombination construct, sacB will be located between the A and B-modules. Successful recombination will cause deletion of the suicide gene and thus cells that contain plasmids with positive recombination events can be selected on sucrose containing media. The sacB gene was amplified using primer MS26 and MS27 from the suicide plasmid pNTPS (kindly provided by K. Thormann, MPI Marburg) via PCR. PCR products were separated by agarose gel electrophoresis, extracted from the gel and cloned into [http://partsregistry.org/Part:pSB1C3 pSB1C3] via [http://partsregistry.org/Help:Standards/Assembly/RFC10 Standard Assembly]. Positive clones were verified by restriction enzyme digest and [http://partsregistry.org/cgi/partsdb/dna.cgi?part_name=BBa%20K866003 DNA sequencing]. [http://partsregistry.org/Part:BBa_K866003 BBa_K866003] can be fused to promoters of different strength and tested for optimal counter-selection through cell death on sucrose containing media.

Experiments:

Generation of test constructs:

As a first step towards our complete recombination construct, we generated the final recombination gene products i.e. the fusion of a complete A-module with a complete B-module connected by a gix recombination site. These putative recombination constructs would be the outcome if a recombination event would have occurred in the complete recombination construct. For this purpose, all three cellular localization domains (A-Modules), HU, GroES and AmiC have been synthesized. The A modules each consist of the localization domain (AmiC, GroES or HU) preceded by a standard ribosome binding site (RBS) [http://partsregistry.org/Part:BBa_J61100 BBa_J61100] and the strong constitutive promotor [http://partsregistry.org/Part:BBa_J23108 BBa_J23108]. Downstream of the localization domains, a gix site is located. Between promotor, RBS, localization domain and the gix site, the [http://partsregistry.org/Help:Standards/Assembly/RFC10 scar sequence] from standard assembly was included to test for the correct expression of the reading frame. The complete A-modules are flanked by the biobrick standard assembly [http://partsregistry.org/Help:Standards/Assembly/RFC10 RFC10] prefix and suffix.

The complete A-modules coding for AmiC and GroES were cloned N-terminally to our fluorescent fusion protein sequences [http://partsregistry.org/Part:BBa_K866000 BBa_K866000] (CFP-Fusion Brick), [http://partsregistry.org/Part:BBa_K866001 BBa_K866001] (mRFP-Fusion brick as well as the biobrick [http://partsregistry.org/Part:BBa_K125500 BBa_K125500] (GFP-Fusion Brick) via standard assembly. Verification of fluorescence was performed via fluorescence microscopy.

Fluorescence microscopy:

In order to screen for fluorescent E. coli harboring our putative test constructs, positively selected colonies through antibiotic resistance have been subjected to fluorescence microscopy. For this purpose, colonies growing on LB-Agar supplemented with antibiotics for [http://partsregistry.org/Part:BBa_K866000 BBa_K866000] (CFP-Fusion Brick), [http://partsregistry.org/Part:BBa_K866001 BBa_K866001] (mRFP-Fusion brick) and [http://partsregistry.org/Part:BBa_K125500 BBa_K125500] (GFP-Fusion Brick), respectively, were cultured over-night in LB-medium with appropriate antibiotics. Small aliquots (10 µL) of overnight cultures were added onto 2% agarose-pads casted on microscope slides, covered by cover-slips and used for fluorescence microscopy.

Altogether, 6 different constructs have been analyzed for fluorescence, being AmiC-GFP, AmiC-CFP, AmiC-mRFP, GroES-GFP, GroES-CFP and GroES-mRFP. As can be seen in Figure 1, fluorescence was observed in all cells carrying a fusion construct. Especially in cells expressing GroES-GFP and AmiC-GFP (Fig. 1 E and B), intense fluorescence was detectable at cell poles for GroES and in the periplasm for AmiC. Similar localizations were also observed for GroES-mRFP and AmiC-mRFP (Fig. 1 D and A). For GroES-CFP and AmiC-CFP (Fig. 1 F and C), fluorescence is detectable, yet for AmiC-CFP no precise localization could be determined.

As a result, our test constructs representing a putative recombination event of our planned recombination construct were successfully expressed in E.coli. Furthermore, GFP- and mRFP fused A-modules show enhanced fluorescence at cell poles for GroES and in the periplasm for AmiC.


Syn 8.jpg


Syn MR Kontrolle (P-RBS).jpg

Fig. 1: Fluorescent E. coli cells harbouring the fusion constructs of A- and B-modules. A: AmiC-mRFP. B: AmiC-GFP. C: AmiC-CFP. D: GroES-mRFP. E: GroES-GFP. F: GroES-CFP. G: E. coli harboring a Promotor-RBS construct which serves as a non-fluorescent control. All images were taken by fluorescence microscopy at a magnification of x100.